PROTEOMIC ANALYSIS OF THE NUCLEOCYTOPLASMIC TRANSPORT MACHINERY

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 核孔复合体 (NPC) 调节真核细胞细胞质和核质之间蛋白质和 RNA 的重要流动。它是一种超分子筛，横跨核膜的两层膜，形成四十纳米的水通道，连接细胞的细胞质和核质。 小代谢物通过 NPC 自由扩散，但大蛋白质和 RNA 的流动是有选择性的，需要货物中的特定运输信号。 这些信号被称为核转运蛋白（也称为输入蛋白、输出蛋白和转运蛋白）的移动受体识别，该受体与 NPC 蛋白（核孔蛋白；Nups）相互作用，在细胞质和核质之间易位和穿梭货物。 我们使用基于 GST 的亲和捕获测定与捕获蛋白质的质谱鉴定相结合，获得了酿酒酵母核质转运机制 54 个组件之间的蛋白质相互作用图谱。 该方法能够识别酵母提取物中的数十种蛋白质，这些蛋白质与运输机械的分离组件（Nups、Kaps、Ran 及其效应器）特异性相互作用。 我们目前正在研究如何在不同的细胞条件下（例如不同的细胞密度或温度）以及细胞在细胞周期的不同阶段中调节相互作用网络。 该方法提供了核细胞质运输分子动力学的全局图，以及该机制的每个蛋白质成分所产生的许多相互作用的详细图谱。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The nuclear pore complex (NPC) regulates the vital flow of proteins and RNA between the cytoplasm and the nucleoplasm of eukaryotic cells. It is a supramolecular sieve that spans both membranes of the nuclear envelope to create a forty-nanometer aqueous channel that connects the cytoplasm and the nucleoplasm of cells. Small metabolites diffuse freely through the NPC, but the flow of large proteins and RNA is selective and requires specific transport signals in the cargo. The signals are recognized by mobile receptors termed karyopherins (also called importins, exportins and transportins), which interact with proteins of the NPC (nucleoporins; Nups) to translocate across and shuttle cargo between the cytoplasm and the nucleoplasm. We are obtaining a map of protein interactions between the 54 components of the nucleocytoplasmic transport machinery in S. cerevisiae using a GST based affinity-capture assay coupled to mass spectrometric identification of captured proteins. The approach has allowed the identification of dozens of proteins in yeast extracts that interact specifically with isolated components of the transport machinery (Nups, Kaps, Ran and its effectors). We are currently examining how the network of interactions is modulated under varying cellular conditions, such as different cell densities or temperature, and as cells progress through different stages of the cell cycle. The approach is providing a global picture of the molecular dynamics underlying nucleocytoplasmic transport, as well as a detailed map of the many interactions that each protein component of the machinery makes.